Quantification of Protein Glycosylation Using Nanopores

Although nanopores can be used for single-molecule sequencing of nucleic acids using low-cost portable devices, the characterization of proteins and their modifications has yet to be established. Here, we show that hydrophilic or glycosylated peptides translocate too quickly across FraC nanopores to be recognized. However, high ionic strengths (i.e., 3 M LiCl) and low pH (i.e., pH 3) together with using a nanopore with a phenylalanine at its constriction allows the recognition of hydrophilic peptides, and to distinguish between mono- and diglycosylated peptides. Using these conditions, we devise a nanopore method to detect, characterize, and quantify post-translational modifications in generic proteins, which is one of the pressing challenges in proteomic analysis.


FraC monomer expression and purification
Plasmid containing the FraC gene was transformed into BL21(DE3) cells using electroporation.
The transformed cells were grown overnight at 37 ºC on LB agar plates supplemented with 1% glucose and 100 µg/ml ampicillin. On the next day, the colonies were pooled together and resuspended and grown in 200 mL 2YT medium at 37 ºC until the OD600 reached a value of 0.6-0.8. At this point, the expression was induced by the addition of 0.5 mM IPTG and the culture was incubated overnight at 25 ºC. Afterwards, the cells were pelleted by centrifugation at 4000 rpm for 15 minutes and the cell pellets were stored at -80 ºC for at least 30 minutes.
For protein purification, cell pellets of 100 ml culture were resuspended in 20 ml lysis buffer,

Single-channel recordings
Two fluidic compartments are separated by a polytetrafluoroethylene (Teflon) film (Goodfellow Cambridge Ltd) with a thickness of 25 µm, containing an aperture of approximately 100 µm in diameter. First, 10 µl of a 5% hexadecane solution in pentane is applied to the aperture and the pentane is left to evaporate shortly. Afterwards, both compartments are filled with 400 µl buffer and 10 µl of a 10 mg/ml DPHPC solution in pentane is added on top of the buffer solution.
The chamber is left to evaporate the pentane and an Ag/AgCl electrode is placed in each compartment as to make contact with the buffer solution. Planar lipid bilayers were formed by repeatedly lowering and raising the buffer solution until a stable lipid bilayer was formed. FraC nanopores were added to the cis-compartment and the lipid bilayer was reformed until a single channel was present. Presence of a single channel and its orientation were confirmed by the IV characteristics of the pore. A two-minute blank was recorded and afterwards substrate was added to the cis-compartment.

Data acquisition
The ionic current was recorded using a Digidata 1440A (Molecular Devices) connected to an Axopatch 200B amplifier (Molecular Devices). All data is recorded with a sampling frequency of 50 kHz and with a Bessel filter of 10 kHz. The data is then digitally filtered using a 5 kHz Gaussian low-pass filter prior to the event detection.

Event detection
First, using Clampfit software, the full-point histogram of the ionic current trace was taken in order to determine the open pore current (Io) and the open pore noise ( 0 ). A Gaussian around the open pore current was fitted to determine the peak centre (Io) and standard deviation ( 0 ).
Then, events were detected using a threshold search with a threshold of 5* 0 and with a minimum duration of 50 µs. The excluded current percent (Iex%) was calculated using % = ( ∆ 0 ) * 100%, where ΔIB (=IO -IB) is the magnitude of the current blockade.

Glycopeptide dwell time analysis
After event detection, the average dwell time of the peptides was estimated for each salt concentration in triplicates. First, the Iex% range was determined for each peptide cluster: 71 to 78% for 9mer_2Glc, 62 to 68% for 9mer_1Glc and 56 to 61% for 9mer_unmod. A log-normal distribution was fitted through the dwell time histogram to obtain the mean dwell time of each peptide cluster. The standard deviation is calculated between the three individual measurements in different nanopores.
Reaction mixtures (total 5.4 mL) consisted of 0.5 mM 9mer peptide (~2.5 mg) (from 10 mM DMSO stock to keep DMSO content low) and 2.5 mM UDP-Glc in the reaction buffer (50 mM HEPES, 100 mM NaCl, 10% glycerol, pH 7.5). Reactions were initiated by the addition of 10 µM ApNGT 2 and incubated at room temperature overnight. The glycosylation of ANVTLNTTG was pushed over the next five days by addition of total extra 7.3 mg of UDP-Glc and 10 µM of ApNGT until ~90% conversion to the di-glycosylated product was reached. To prepare the samples for LCMS analysis, an aliquot of the reaction mixture was diluted 10-fold and analyzed with RP-LCMS (1 µL injection, Acquity UPLC HSS T3 column (Waters, 2.1×150 mm, 1.8 µm) in combination with eluents A (0.1% formic acid in H2O) and B (0.1% formic acid in acetonitrile), 20 min run (flow rate 0.3 mL/min) with a linear gradient from 5% to 95% of B in 13 min with subsequent increase to 95% B for 3min and return to 5% B). Before anion exchange purification, reaction mixtures were filtered through a 0.2 µm filter. The resulting peptides were separated from the ApNGT, UDP and UDP-Glc by strong anion exchange on FPLC ÄKTA system (GE Healthcare). For this, 1 mL of the reaction mixture (~ 0.2 mM peptide concentration) was applied on a Q FF column (5 mL, GE Healthcare) with a flow rate of 1.5 mL/min. The column was eluted with the linear gradient from 0 to 60% Buffer B (0.9 M NH4HCO3) in ten column volumes, with subsequent increase to 100% in four CV. Elution was monitored with UV (214 nmpeptide and 280 nm -UDP, UDP-Glc). The fractions containing glycosylated peptides (first 5-6 min of the elution) were pooled and freeze-dried. Dried samples were reconstituted in DMSO to prepare stocks for the nanopore analysis.

Rhamnosylation and purification of the 11mer_Pa peptide
To prepare rhamnosylated 11mer-L-Pro-D-Pro_Pa, 2.7 mM 11mer_Pa (1 mg), 6.1 mM TDP-Rha (1.2 mg) and 41 µM EarP were incubated at room temperature for two days in the reaction buffer (20 mM Tris, 100 mM NaCl, pH 8). The reaction was pushed to full conversion over the next three days by addition of total extra 1 mg of TDP-Rha and 24 µM of EarP until ~90% conversion was reached. Subsequently, the reaction mixture was diluted to a 0.5 mM concentration of Rha-11mer, applied to an Amicon spin filter (MWCO 10 kDa, 15 mL,Millipore) and centrifuged at 5000 x g to remove the enzyme. The resulting solution was further purified from TDP and TDP-Rha by strong anion exchange on FPLC (ÄKTA system, GE Healthcare).
For this, 0.25 mL of the reaction mixture was applied on Q FF column (5 mL, GE Healthcare) with flow rate 1 mL/min in five column volumes (CV). The column was eluted with the linear gradient from 0 to 10% Buffer B (1M NH4HCO3) in two CV with subsequent increase to 100% in four CV. Elution was monitored with UV (214 nmpeptide and 280 nm -TDP, TDP-Rha).
The fractions containing rhamnosylated 11mer_Pa peptide were pooled and freeze-dried.
Residual buffer salts were removed by desalting with PD-10 desalting columns (GE Healthcare). Desalted fractions of Rha-11mer were freeze-dried and aqueous stock of 0.5 mM was prepared for the nanopore studies.

Quantification of rhamnosylation on peptides using the nanopore
After event detection, the Iex% spectra of the three measurements were first realigned to correct for small shifts in the baseline. After baseline correction, a Gaussian mixture model was used to detect the event clusters. We found that the clusters are best detected in the Iex% vs event noise (ISD) spectrum, where ISD is the fluctuation of the ionic current during the event. From the fitting of the Gaussian mixture model, the location and distribution of the event clusters in the Iex% vs ISD spectrum was obtained. For each detected event cluster, the center (µ1) and the spread (σ1) in Iex% and the center (µ2) and spread (σ2) in ISD was determined. Then, for each event cluster the events that satisfy both (µ1-σ1) > Iex% > (µ1+σ1) and (µ2-σ2) > ISD > (µ2+σ2) were counted to obtain the number of events belonging to the rhamnosylated peptide (nRha) and the unmodified peptide (nunmod). In addition, the events that satisfy the same equation in the blank measurement were subtracted to reduce the effect of intrinsic current blockages. The percentage of rhamnosylation in the sample is estimated as: nRha/(nRha+nunmod)*100%. These values are then used in equation 2 to calculate the RDF. Finally, the conversion is calculated using: The standard deviation is calculated between the three individual measurements in different nanopores.

Rhamnosylation and purification of EF-P
To prepare rhamnosylated EF-P, 12 μM of EF-P (after His6-SUMO-tag cleavage, as described in 3 ) was incubated with 2 μM of EarP-His6-SUMO and 100 μM TDP-Rha in the reaction buffer (20 mM Tris, 500 mM NaCl, pH 8) overnight at room temperature. The next day, Ni-affinity chromatography was used to isolate Rha-EFP. Briefly, the reaction mixture was incubated with Ni-NTA resins for 1.5 h at 4 ºC with gentle shaking. The resulting suspension was allowed to pass through the gravity column and the flow-through was collected. Next, the resin was washed once with lysis buffer (20 mM Tris, 500 mM NaCl, pH 8), followed by washing buffer 1 in two steps (20 mM Tris, 500 mM NaCl, 15 mM imidazole, pH 8), and washing buffer 2 in two steps (20 mM Tris, 500 mM NaCl, 30 mM imidazole, pH 8). This was followed by elution with elution buffer (20 mM Tris, 500 mM NaCl, 400 mM imidazole, pH 8). Analysis of the purification fractions with SDS-PAGE ( Figure S7) indicated that Rha-EFP was present in the flow-through, lysis buffer wash, and washing buffer (1 and 2) fractions. To prepare the Rha-EF-P sample for the nanopore analysis, fractions containing the protein were pooled, diafiltrated, and concentrated with Amicon spin filter to 6.5 mg/mL (0. 3 mL total). The purity of the sample was confirmed with intact protein MS analysis ( Figure S8).

Lys-C digestion of EF-P and rhamnosylated EF-P
200 ng of protein was dissolved in 180 µl buffer containing 100 mM Tris, buffered to pH 8.0.
Then we add 4 µg of Lys-C, yielding a 1:50 enzyme : protein mass ratio and the sample is subsequently incubated overnight at 37 ºC. On the next day, an Amicon filter with a molecular weight cut-off of 3000 Da was used, to eliminate the protease and any undigested protein from the sample. The sample is stored at -20 ºC until use.   Figure S4: Initial screening of other FraC mutants for glycopeptide detection. Ionic current traces (left) and event characteristics (right) of measurements in FraC mutants with single glycopeptides. Top panel: 30 µM of 9mer_2Glc added to a FraC D10R nanopore, measured at +100 mV applied voltage. Middle panel: 10 µM of 9mer_2Glc added to a FraC G13H nanopore, measured at +50 mV applied voltage. Bottom panel: 7.5 µM of 9mer_2Glc added to a FraC G13W nanopore, measured at -70 mV applied voltage.
20mdv048-YAK-EFP_XT_00001_MHp__200608123306 #1 RT: 1.   The -10lgP score relates to the probability of detection and the peak area (Area EF-P) relates to the concentration of the peptides in the sample. The second column shows the peptide numbers according to Table 2. The -10lgP score relates to the probability of detection and the peak area (Area Rham EF-P) relates to the concentration of the peptides in the sample. The second column shows the peptide numbers according to Table 2.